A method for the generation of combinatorial antibody libraries using pIX phage display

Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):12612-6. doi: 10.1073/pnas.192467999. Epub 2002 Sep 18.

Abstract

For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and select a wide variety of antibodies. We previously reported that the phage coat proteins pVII and pIX could be used to display the heterodimeric structure of the antibody Fv region. Herein, aspects of this technology were invoked and extended to construct a large, human single-chain Fv (scFv) library of 4.5 x 10(9) members displayed on pIX of filamentous bacteriophage. Furthermore, the diversity, quality, and utility of the library were demonstrated by the selection of scFv clones against six different protein antigens. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning. Analyzed scFvs were also found to be of high affinity. For example, kinetic analysis (BIAcore) revealed that scFvs against staphylococcal enterotoxin B and cholera toxin B subunit had a nanomolar and subnanomolar dissociation constant, respectively, affording affinities comparable to, or exceeding that, of mAbs obtained from immunization. High specificity was also attained, not only between very distinct proteins, but also in the case of the Ricinus communis ("ricin") agglutinins (RCA(60) and RCA(120)), despite >80% sequence homology between the two. The results suggested that the performance of pIX-display libraries can potentially exceed that of the pIII-display format and make it ideally suited for panning a wide variety of target antigens.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacteriophages / chemistry*
  • Bacteriophages / genetics
  • Bacteriophages / metabolism
  • DNA, Complementary / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Variable Region / genetics
  • Kinetics
  • Peptide Library*
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • DNA, Complementary
  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Peptide Library
  • Recombinant Proteins
  • Tumor Necrosis Factor-alpha